The main goal of the research presented within this proposal is to further our understanding of the molecular mechanisms of how the tumor suppressor retinoblastoma (Rb), regulates transcription via interactions with E2F family members. Several genes important for cell cycle progression and DNA synthesis have been identified as E2F targets. However, few experiments have been performed using natural promoters, and studies of E2F transcriptional regulation have focused primarily on synthetic promoter constructs as naked DNA templates. Therefore, using primer extension, and footprinting assays, the mechanisms of E2F transcriptional regulation will be investigated both on naked DNA and on reconstituted chromatin templates using several natural promoters as models. These will include the cyclin E (c-myc and B-myb) promoters each of which is suspected to be under the control of E2F and Rb family members. These experiments should help to address the molecular mechanisms used by Rb to regulate transcription from a natural promoter and in the context of a chromatin environment. Also proposed are studies to identify chromatin-remodeling factors recruited to the cyclin E promoter. Such experiments are based upon a recent link between Rb and histone deacetylases. These studies will add to our understanding of Rb's normal function which when perturbed can contribute to retinoblastoma and other human cancers.